Götz Baumann

Functional analysis of sequences required for transcriptional activation of a soybean heat shock gene in transgenic tobacco plants.

The 5′ DNA sequences involved in the thermal inducibility of the soybean heat shock gene hs6871 were analysed in transgenic tobacco plants. The transcriptional activity of various in vitro generated deletion mutants was examined by Northern blot analysis, S1 nuclease mapping and dot‐blot hybridization. At least 181 bp upstream from the translational start site are sufficient for thermal induction at 40°C and correct initiation of transcripts. Full promoter activity with the induction of wild‐type levels of transcripts requires additional upstream sequences contained within 439 bp 5′ to the coding sequence. Our results suggest that faithful regulation and the generation of high levels of hs6871‐specific mRNA depend on the presence of sequences which show homology to the 14‐bp heat shock consensus element of Drosophila and, in addition, on as yet unidentified enhancer‐like upstream sequences.

The function of plant heat shock promoter elements in the regulated expression of chimaeric genes in transgenic tobacco

SummaryA series of deletion mutants of a soybean heat shock (hs) gene promoter was generated and linked to the chloramphenicol acetyl transferase (CAT) coding sequence. These chimaeric promoter/reporter gene constructs were introduced into tobacco and thermoregulated expression of CAT activity was examined in leaf extracts. Three different types of gene fusions were tested using two differentBIN19 vector constructions: (1) translational fusion between the N-terminus of the protein coding sequence of the heat shock geneGmhsp17.3-B and CAT; (2) transcriptional fusions between the 5′ nontranslated RNA regions ofGmhsp17.3-B and CAT; and (3) promoter fusions joining the hs promoter upstream sequences to the TATA box sequence of the Δ CaMV 35S-CATter vector. Alternatively, multiple copies of a synthetic deoxyoligonucleotide with the soybean hs consensus element (HSE2) were used. Heat inducible CAT activities were detected except in plants containing a transcriptional fusion devoid of all but 18 nucleotides at the 5′ terminus of the hs gene transcript. CAT activity was detectable in these plants only during the recovery at 25° C after a hs (40° C). Overlapping HSE-like promoter sequences seem to be necessary for the induction of heat inducible transcription of linked genes; synthetic HSE2 sequences have the capacity to reconstitute a hs promoter in combination with a TATA box sequence. Effective translation during hs seems to require sequences in the 5′ nontranslated leader of the hs protein mRNA; these sequences can be functionally replaced by the 5′ leader sequence of the Δ CaMV 35S promoter.

Rapid identification of phosphopeptide ligands for SH2 domains. Screening of peptide libraries by fluorescence-activated bead sorting.

A method for the identification of high-affinity ligands to SH2 domains by fluorescence-activated bead sorting (FABS) was established. Recombinant SH2 domains, expressed as glutathione S-transferase (GST) fusion proteins, were incubated with a phosphotyrosine (Y*)-containing peptide library. 6.4 × 105 individual peptides of nine amino acids in length (EPX6Y*X19X7X19X7X6) were each displayed on beads. Phosphopeptide interaction of a given SH2 domain was monitored by binding of fluorescein isothiocyanate-labeled antibodies directed against GST. High-fluorescence beads were isolated by flow cytometric sorting. Subsequent pool sequencing of the selected beads revealed a distinct pattern of phosphotyrosine-containing motifs for each individual SH2 domain: the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence Y*ENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk selected Y*EELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine. For deconvolution studies, soluble phosphopeptides comprising variations of the Grb2 motifs were resynthesized and analyzed by surface plasmon resonance.

Thermo-induced transcripts of a soybean heat shock gene after transfer into sunflower using a Ti plasmid vector

A genomic DNA fragment containing a soybean heat shock gene (hs6871) was inserted into the T‐DNA region of the Agrobacterium tumefaciens pTiC58 plasmid. A strain carrying the modified Ti plasmid was used to incite tumors in sunflower hypocotyls. The expression of the heat shock gene was investigated by Northern blot analysis of RNA and S1 nuclease mapping of the transcriptional start site. Heat shock‐induced poly(A) mRNA was detected in tumor tissue only after incubation at 40°C (heat shock) not at the normal growth temperature (28°C). Transcripts from hs6871 are faithfully initiated in sunflower, starting at the same site on the DNA as in soybean. The low level of transcripts initiating correctly on hs6871 in sunflower is consistent with a general tissue‐specific reduction in the expression of partially homologous native heat shock genes in sunflower tumors.

The Expression of Heat Shock Genes — A Model for Environmental Stress Response

When cells, tissues, or whole organisms are subjected to grossly elevated temperatures or heat shock (hs), they respond transiently by synthesizing a number of new proteins, the prevalent heat shock proteins (hsps). Hsps are operationally defined as those proteins whose synthesis is immediately and dramatically induced at high temperatures. The induction of hsp synthesis is the major feature of the hs response; other aspects of the reprogramming of cellular activities are less well understood (for recent reviews see Burdon, 1986; Lindquist, 1986; Nagao et al., 1986; Schlesinger, 1986; Schoffl et al., 1986). Some of the general characteristics relevant to plants are briefly summarized here.